title: Purification of integral membrane proteins and lipid-binding assays.
abstract: The lipid droplet (LD) is an evolutionarily conserved organelle composed primarily of triglycerides (TAG) and cholesteryl esters. Recently, Fat storage-Inducing Transmembrane proteins 1 & 2 (FITM1/FIT1 and FITM2/FIT2) were discovered as a conserved family of proteins involved in fat storage. FIT1 and FIT2 are both localized to the endoplasmic reticulum, but have distinct tissue distributions. FIT proteins mediate TAG LD accumulation when overexpressed, but do not synthesize TAG. FIT proteins function by partitioning newly synthesized TAG into LDs. In order to understand the mechanism by which this occurs, a method was developed to purify FIT proteins from insect cells in detergent micelles. The ability of purified FIT proteins to bind TAG and other neutral lipids was tested in detergent micelles, demonstrating lipid specificity and saturation binding. These techniques can be applied to a variety of proteins in lipid biology in an effort to try to reconstitute a mechanism of action or protein activity. The methods that will be discussed here can also be scaled to either screen a library of mutant proteins for binding to a particular compound or utilized to delineate structural requirements of ligands that are important for protein-ligand interactions. Here, we present a detailed description of the purification protocol and micellar protein-ligand binding experiments and their possible applications.
authors.affiliation: Biochemistry Department, Albert Einstein College of Medicine, Bronx, New York, USA; Program in Cardiovascular & Metabolic Disorders, Duke-NUS Graduate Medical School Singapore, Singapore.
authors.forename: David A
authors.initials: DA
authors.lastname: Gross
authors.forename: David L
authors.initials: DL
authors.lastname: Silver
mesh_terms: D017667:Adipocytes / Q000378:metabolism; D004721:Endoplasmic Reticulum; D006801:Humans; D002479:Inclusion Bodies / Q000737:chemistry / Q000378:metabolism; D008024:Ligands; D050356:Lipid Metabolism; D008055:Lipids / Q000737:chemistry; D008565:Membrane Proteins / Q000302:isolation & purification* / Q000378:metabolism; D008823:Micelles; D011485:Protein Binding; D014280:Triglycerides / Q000737:chemistry / Q000378:metabolism
chemical_list: C572548:FITM1 protein, human; C575964:FITM2 protein, human; D008024:Ligands; D008055:Lipids; D008565:Membrane Proteins; D008823:Micelles; D014280:Triglycerides
keywords: Adipocytes; Detergent micelles; Diabetes; Diacylglycerol; FIT; Lipid droplets; Obesity; Triglyceride